Determination of levels of immunogenic gluten peptides in human samples

ABSTRACT

The present invention, fitted in the medical-clinical sector, shows a method for monitoring the ingestion of gluten by measuring protein/gluten peptides present in fecal samples with antibodies against immunogenic peptides resistant to gastrointestinal digestion. The presence or absence of said immunogenic peptides is controlled by immunological assays based on reactive antibodies against immunogenic gluten peptides that are resistant to proteolysis. These assays may be quantitative techniques as ELISAs, or qualitative as rapid immunochromatographic assays, immunoblots, etc. These measures may also be applied to verify compliance with the gluten-free diet, to improve diagnosis in cases of refractory or severe symptoms of celiac disease, in cases in which a gluten-free diet is supposedly being respected, or to clinical research on the effectiveness of enzymatic therapies related with prolamin detoxification.

FIELD OF THE INVENTION

The present invention, fitted in the medical-clinical sector, shows amethod for monitoring the ingestion of gluten by measuringprotein/gluten peptides present in fecal samples with antibodies againstimmunogenic peptides resistant to gastrointestinal digestion. Thepresence or absence of these immunogenic peptides is controlled byimmunological assays based on antibodies reactive against immunogenicgluten peptides that are resistant to proteolysis. These assays may bequantitative techniques ELISAs, or qualitative as rapidimmunochromatographic assays, immunoblots, etc. These measures may alsobe applied to verify compliance with the gluten-free diet, to improvediagnosis in cases of refractory or severe symptoms of celiac disease inwhich a gluten-free diet is supposedly being respected, or to clinicalresearch on the effectiveness of enzymatic therapies related withprolamin detoxification.

BACKGROUND OF THE INVENTION

Gluten is a set of storage proteins of cereals. Gluten proteins fromwheat, barley, rye and probably oats, are not tolerated by geneticallypredisposed individuals with celiac disease (CD). In wheat, gluten iscomposed of an ethanol-soluble fraction (prolamins: α, β, γ andω-gliadins) and other insoluble, glutenins (high and low molecularweight subunits) (Wieser, 2007 Food Microbiol., 24:115-119; Fasano,2009, Sci Am, 301:54-61). Gliadins and glutenins are also unusually richin proline (˜15%) and glutamine (˜35%) residues. As a result, while mostdietary proteins are digested by gastrointestinal proteases to singleamino acids, dipeptides or tripeptides, gluten proteins are notcompletely digested (Erickson and Kim, 1990, Annu Rev Med 41:133-139,Gray, 1991, New York: Oxford University, pp. 411-420; Ganapathy et al.,2006, Academic Press, pp. 1667-1692). Therefore, some of the glutenpeptides generated during gastrointestinal digestion are highlyresistant to digestion by gastric and pancreatic enzymes, so that theypersist in the gut. These peptides are capable of being internalizedinto the intestinal cells and, therefore, glutamine residues can bedeaminated by tissue transglutaminase (tTG). Genetic predisposition ofindividuals with CD makes them intolerant to these peptides becausetheir immune system reacts pathologically against autoantigens generatedby gluten peptides/tTG-interaction (Korponay-Szabó et al., 2007, BMJ,335:1244-1247; Bethune and Khosla, 2008, PLoS Pathogens, 4: e34; Jabriand Sollid, 2009, Nat Rev Immunol., 9:858-870). Deamidated peptidesinduce an immune response mediated by T cells that causes chronicinflammation of the small intestine. Intestinal villi are destroyed dueto the immunological reaction, resulting in a reduction of theintestinal absorption which can lead to symptoms such as diarrhea,anemia, stunting, weight loss, bone disorders, neurological disorders,cancer, etc. (Alaedini and Green, 2005, Ann Int Med, 142:289-299;Catassi and Fasano, 2008, Curr Opin Gastroenterol., 24:687-691; Tack etal., 2010, Gastroenterol Hepatol., 7:204-213).

One of the main gluten peptides described to date is the 33-mer peptidefrom α2-gliadin (Shan et al., 2002, Science, 297:2275-2279; Bethune etal., 2009, Chem Biol, 16:868-881) that has been shown to be resistant togastrointestinal digestion, substrate of the tTG mediated deaminationand highly reactive with T cells isolated from celiac patients. Theidentification of the 33-mer peptide and other peptides, helps todemonstrate that gluten epitopes with high antigenicity are located ingliadin regions rich in proline and glutamine residues (Shan et al.,2002, Science, 297: 2275-2279; Tye-Din et al., 2010, Sci Transl Med 2:41RA51).

Nowadays, the only existing therapy for patients with celiac disease isa strict gluten-free diet (GFD). Non-compliance with the GFD has beenassociated with osteoporosis, iron deficiency anemia, depression andinfertility, all of which is improved, to some extent, by adhering tothe gluten-free diet. These observations give us an idea of theimportance of adherence to a GFD to reduce symptoms, prevent nutritionaldeficiencies and improve the quality of life of these patients. However,several studies based on intestinal biopsies have suggested that dietarytransgressions are relatively frequent, being between 32.6% and 55.4% inthe populations studied (Ciacci et al., 2002, Digestion, 66: 178-185;Sylvester and Rashid, 2007, Can J Gastroenterol., 21:557-564). The lackof adherence to a strict gluten-free diet is the main reason for poorlycontrolled celiac disease in adults.

In addition, there is a part of the celiac population that does not seemto respond positively to the GFD and suffer symptoms of persistent orrecurrent malabsorption and intestinal villous atrophy. This populationcould be suspected of having refractory CD, a rare disease(approximately 5%-10% of patients with CD) that appears in patientswithout apparent positive response to the gluten-free diet (Al-Shot etal., 2007, Dig Dis 25:230-236, Freeman, 2009, Gut Liver, 3:237-246;Rubio-Tapia and Murray, 2010, Gut, 59:547-557). Although this refractorydisease was described in patients with assumed total absence of glutenintake, involuntary ingestion and hypersensitivity to a small amount ofgluten can also trigger the symptoms of the disease. The lack of anaccurate marker for monitoring compliance with the GFD is still anunresolved issue and is particularly difficult in the case of minordietary transgressions (Fernandez-Calle et al., 1993, Gut, 34:774-777).There is no way to demonstrate gluten intake and thereby avoid possibleharmful consequences. In fact, the consequences of dietarytransgressions can only be measured by observing mucosal inflammationand/or villous atrophy for which intestinal biopsies would have to beperformed and, as a result, the patient would have to be anesthetisedwith the possible consequences that this may have.

Control of anti-tTG has been proposed as a marker to assess the strictcompliance with the GFD. However the effectiveness of this marker tocontrol the intake of gluten is not yet clear (Tack et al., 2010,Gastroenterol Hepatol., 7:204-213). Other markers have been proposed formonitoring the diet, such as permeability test (Duerksen et al., 2005)or fecal calprotectin (Ertekin et al., 2010, J Clin Gastroenterol.,44:544-546). These methods can demonstrate the presence of inflammatoryprocesses, so that if the values of these markers are altered it can bea result of infectious diseases, inflammatory bowel diseases or allergyprocesses, meaning that they do not need to be a measure of the directintake of gluten. Therefore, there is no effective method to verify thatthe celiac patient is performing a GFD or to eliminate the possibilitythat the refractory CD symptoms are due to a hypersensitive intoleranceto gluten traces associated with unintentional exposure to toxiccereals.

Compliance with the diet assessed by interview has been suggested as amarker of CD control for its low cost, non-invasiveness, and its provencorrelation with intestinal damage. However, GFD involves numerousrestrictions for patients because of its social and economicimplications. Additionally, a gluten-free diet is difficult to maintaindue to the ubiquity of gluten in foods, educational misinformation,changes in food labeling and possible cross-contamination in food(Bethune et al., 2009, Chem Biol, 16:868-881; Selimoglu and Karabiter,2010, J Clin Gastroenterol., 44:4-8). Moreover, certain lifestyles andsome sectors of the population make difficult, to some extent,compliance with the GFD. Furthermore there is no alternative to patientinterviews to know how reliable the results of these clinical trialsare.

A more direct measure of the ingestion of gluten could provide criticalinformation about the patient: the detection of infringements of the GFDbefore anatomical damage, inadvertent consumption detection, theaccuracy assessment of the adherence to treatment in the initial periodafter diagnosis when patients are less familiar with the diet, etc.providing an easy and reliable confirmation of the results obtained.Therefore, a sensitive and reliable marker to monitor and detect glutenintake could be a useful tool for the proper compliance with the GFD andprobably for an accurate diagnosis of refractory CD.

Monoclonal antibodies (moAbs) G12 and A1 obtained against the mainimmunogenic epitope of α-gliadin have demonstrated to be very useful inthe detection of toxic peptides in food samples as well as in clinicalresearch of gluten enzymatic detoxification (Morón et al., 2008, Am JClin Nutr., 87:405-414; Morón et al., 2008, PLoS ONE, 3: e2294; Ehren etal., 2009, PLoS ONE, 4: e6313, Alvine Pharmaceuticals, Inc., BiomedalSL). The sensitivity and specificity of the monoclonal antibodies andtheir ability to recognize peptides resistant to gastrointestinaldigestion could make them ideal for monitoring immunotoxic glutenpeptides obtained after intestinal digestion in human samples.Recognition epitopes from moAb G12, QP(Q/E)LP(Y/F), are present in majorpeptides described recently in a high throughput screening performedwith 2,700 peptides from prolamins of different cereals (Tye-Din andal., 2010, Sci Transl Med 2:41 RA51). Undigested peptide fragments fromgluten intake that are not absorbed could be recovered from the feces,which would demonstrate gluten intake by the individual.

In this patent, we have evaluated the feasibility of monitoring gluten,intact and digested, in the feces by the detection of epitopesassociated with the 33-mer peptide, which could be used in clinicalstudies and dietary monitoring, as well as in the diagnosis ofrefractory CD.

DESCRIPTION OF THE INVENTION

The gluten-free diet is the only effective treatment today for celiacdisease. Therefore, compliance with GFD should be monitored to preventcumulative direct and indirect damage, as well as to confirm thepersistence of any symptom of celiac disease is not due to atransgression (intentionally or not) of the diet. However, currentlythere are no methods to monitor dietary compliance in patients withceliac disease.

The aim of the present invention is the application of immunologicalmethods for monitoring compliance with the gluten-free diet by detectingimmunotoxic peptides present in feces that are resistant togastrointestinal digestion. It is an object of the invention theapplication of immunological techniques based on antibodies thatrecognize immunogenic peptides from gluten that resist gastrointestinaldigestion. Preferably, the invention employs immunological techniquesusing antibodies recognizing the 33-mer peptide from gliadin. Apreferent way of making the detection of peptides is by rapidqualitative methods based on immunochromatographic strips or byquantitative and automated methods as ELISA techniques. The preferredmethod used in the invention should be able to detect gluten ingestedequivalent to 50 mgs of wheat gluten per day, which is the maximumamount described for a gluten free diet, or at least 20 ppms of glutenequivalent in feces.

It is also an object of the present patent kits or immunologicalanalytical devices based on antibodies reactive against the 33-mer fromgliadin that are suitable for the detection of gluten peptides in feces.

These procedures and analytical kits are also an object of this patentbecause of its use to reveal a lack of adherence to GFD, either due tocontamination of food consumed or to a voluntary/involuntary occasionalintake of foods containing gluten. Furthermore, it is object of thepresent invention the application of the detection of these immunotoxicpeptides in feces for the clinical research control on celiac disease,including enzymatic therapies related to gluten prolaminsdetoxification, seize of immunotoxic peptides and other alternativetherapies.

The object of the present invention is a method for detecting ormonitoring the gluten ingested by detecting immunotoxic peptides infeces, characterized by the use of immunological methods usingantibodies that recognize preferably epitopes related to 33-mer gliadinpeptide and other peptide sequences resistant to gastrointestinaldigestion.

It is an object of the present invention the use of immunologicaltechniques for such gluten monitoring, immunological methods such asindirect ELISA, competitive ELISA, sandwich ELISA, immunochromatographicstrips, fluorescent immunomicroparticles, Western blot, biosensors basedon electrochemical reactions catalyzed by enzymes attached to theantibodies, by magnetic particles coated with antibodies, by surfaceplasmon resonance, and other techniques in which an analyte bound to anantibody is detected.

In a preferred embodiment of the invention, these methods arecharacterized by the preferent use of one or more monoclonal antibodiescapable of detecting epitopes contained or similar to peptide 33-mer(SEQ ID No. 1), such as the following sequences: SEQ ID No. 2, SEQ IDNo. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ IDNo. 8. In a preferred embodiment of the invention the antibodies usedfor immunological techniques would be G12 and A1 because of the provenrelation between its reactivity and the potential immunotoxicity of asample. The invention also contemplates the use of the antibody R5 thatreacts with the epitope SEQ ID No. 9, which also can be found in glutenpeptides resistant to gastrointestinal digestion, but with lessspecificity against immunogenic peptides resistant to proteases.

A preferred embodiment of the invention would be the use ofimmunological methods based on G12 monoclonal antibody conjugated to anenzyme that allows a quantitative assay using chromogenic, luminescentor fluorogenic substrates. This procedure would use a standard ofgliadin, hydrolysed gliadin, complete 33-mer peptide or a part of itssequence of at least 6 amino acids (SEQ ID No 2).

Another object of the invention is constituted of the particular use ofimmunochromatographic strips based on the anti-33-mer from gliadinantibodies, G12 and A1 which allow a rapid and semiquantitativedetection of proteins/peptides from gluten content in feces.

The invention proposes a measure of the gluten ingested by theindividual through the diet. This is a useful process for the control ofthe gluten ingested by using analytical methods in which a correlationbetween the quantity of ingested gluten and the estimated quantity ofgluten protein/peptides in feces obtained from these methods has beenshown.

The invention proposes an analytical instrument for monitoringcompliance with the GFD, as well as to discard uncontrolled intake ofgluten in patients suspected of suffering from the called refractoryceliac disease. Also, with this procedure, new therapeutic alternativesfor enzymatic detoxification of gluten and other alternative therapiesmay also be controlled in the feces of celiac patients subjected toclinical trials or therapeutic prescription in the future, since theeffectiveness of the therapy could be determined by measuring thepresence or absence of peptides in feces after 12-48 hours from theintake of a controlled amount of gluten in conjunction with therapies toeliminate immunotoxic peptides.

Some issues that are not still resolved in clinical practice, such asthe control of the GFD or the control of involuntary exposure to glutendue to food contamination, could be resolved with simple immunologicalassays in feces. Medics, clinicians and analysts might consider thesemethods useful for clinical trial design and monitoring of their celiacpatients to establish consistent conclusions on the state of thepatient's disease.

Extraction of the peptides from the feces can be carried out directlywith a hydroalcoholic solution of 40 to 60%. Sometimes, due to thenature of the food ingested, the gluten extraction from the feces couldbe improved by adding a solution containing dispersing agents such asguanidinium chloride, arginine chloride, etc., or detergents such aspolyvinylpyrrolidone, and reducing agents such as b-mercaptoethanol, DTTor TCEP.

Subsequently extracted gluten polypeptides are diluted in a bufferedsaline solution and are then used to make the measurement with an ELISA,either competitive or sandwich if an idea of the concentration ofreactive peptides is to be obtained. The standard curve could be donewith standard gliadin digested by trypsin and pepsin to simulate gastricdigestion. Synthesized polypeptide reacting with the antibodies couldalso be used directly, preferably 33-mer from gliadin or fragmentsthereof with the option of making some specific modifications thatretain reactivity against the antibodies. For example the peptides thatmay be used for antibody G12 are the following: SEQ ID No. 1, SEQ ID No.2, SEQ ID No. 3, and SEQ ID No. 4. For A1, besides 33-mer, the followingpeptides could be valid to make the standard curve with a competitiveELISA: SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8.

For a qualitative assay in which it is seen if the value of glutenpolypeptides in feces is greater or less than a certain amount,immunochromatographic strips or ELISAs assays, using antibody G12 or A1or both, could be used. The procedure could be carried out with a kitthat contained the extraction solution of the polypeptides in feces; areference pattern with gluten polypeptides hydrolysed by pepsin andtrypsin or synthesized; and the components of an ELISA with a multi-wellplate and using the antibodies A1 and/or G12 to immobilize the wellsand/or for the development of the assay, or immunochromatographicstrips.

FIGURE LEGENDS

To complement the description being made and in order to aid a betterunderstanding of the characteristics of the invention, according to apreferred practical embodiment thereof, it is attached as an integralpart of said description, with illustrative and non limiting character,the following figures:

FIG. 1. Relative affinity of moAb G12 to immunotoxic peptides derivedfrom PWG gliadin after simulating gastrointestinal digestion. A and B.SDS-PAGE and Western blot of PWG gliadin, PWG gliadin+pepsin and PWGgliadin+pepsin+trypsin/chymotrypsin. Samples were stained with silver ortransferred to a PVDF membrane with moAb G12. MW: molecular weightmarker. C. Analysis by competitive ELISA G12-HRP of peptides fromgliadin PWG.

FIG. 2. Resistance of 33-mer peptide to be broken by gastrointestinalenzymes. A. Amino acid sequence of 33-mer peptide. The recognitionsequence of moAb G12 into the 33-mer peptide is in bold. B. CompetitiveELISA for the detection of 33-mer after treatment with pepsin, trypsinand chymotrypsin using the moAb G12-HRP. C. Western blot of 33-merpeptide after treatment with gastrointestinal enzymes. MW: molecularweight marker. Two separate assays with 3 replicates each one werecarried out.

FIG. 3. Gluten detection in feces from healthy individuals subjected toa gluten-controlled diet. A and B. Gluten peptides/proteinssemiquantification in feces of healthy individuals (n=11) byimmunochromatographic strips G12. The upper line is a positive internalcontrol indicates that the device has worked correctly, and bottom lineindicates the presence of gluten. HL901-HL911: subjects who participatedin the study. *Gluten traces were detected. C and D. SDS-PAGE andWestern blot of gluten peptides and proteins extracted from the feces.MW: molecular weight marker.

FIG. 4. Concentration of 33-mer peptide (ng/mg) in feces after agluten-controlled diet. Competitive ELISA G12-HRP to determine therelationship between gluten proteins ingested/excreted by its content in33-mer peptide. The concentration of 33-mer peptide was determined by astandard curve of 33-mer. Two separate tests were carried out, each withthree replicates.

PREFERRED EMBODIMENT OF THE INVENTION EXAMPLE 1 Quantification of ToxicPeptides from Gliadin PWG Obtained after Simulated GastrointestinalDigestion

The present example shows that a substantial portion of the immunogenicpeptides of gluten remain susceptible for fecal detection despitegastrointestinal digestion. Among the major proteins of the diet, thosethat constitute gluten are the only ones that contain approximately 15%of proline residues and 35% of glutamine residues. The high content ofthese two amino acids prevents the complete proteolysis of theseproteins by gastric and pancreatic enzymes, so that peptide fragmentsare formed in the small intestine which are immunotoxic for celiacpatients. In particular, the 33-mer peptide was found as one of the maincontributors to the immunotoxicity of gluten (Shan et al., 2002,Science, 297:2275-2279). This peptide of the α-2 gliadin contains sixrecognition epitopes for T cells and is highly resistant to proteolysis.

The moAb G12 is specific for the epitope of six amino acids SEQ ID No.10, with 3 repetitions in the 33-mer peptide. Moreover, this antibody iscapable of recognizing other immunoreactive peptides present in gliadinand other toxic prolamins. The purpose of this example is to know thecapacity of G12 antibody to detect toxic peptides formed aftergastrointestinal digestion simulation of gliadin. For standardization ofthe assay PWG gliadin was used, considered an international referencereagent in gluten analysis due to its high content of gliadins, goodsolubility, homogeneity, stability and for being constituted of 28 wheatEuropean cultivars (Eckert et al., 2006, J Cereal Sci, 43:331-341).

Gliadin was subjected to sequential digestion with pepsin (main proteasepresent in the stomach), trypsin and chymotrypsin (proteases containedin the intestinal membrane). The samples were incubated at 37° C. in HClsolution (pH 2) containing 0.06 mg/mL of pepsin. Samples were incubatedfor 60 min and inactivated by heating at 95° C. for 5 min. Aftersimulating gastric digestion with pepsin, the digestions were adjustedto pH 6.0 with sodium phosphate buffer, and incubated with pancreaticproteases: trypsin (0.375 mg/mL) and chymotrypsin (0.375 mg/mL). Afterthe duodenal simulation at 37° C. for 30 min the samples wereimmediately inactivated at 95° C. for 5 min.

The proteic profile of the prolamins fractions which constitute PWGgliadin was analyzed by SDS-PAGE to observe the pattern of bandsobtained after the enzymatic treatment and to confirm that samples hadbeen digested. For the analysis by SDS-PAGE, the samples were diluted inrunning buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 0.001%bromophenol blue and 5% 2-mercaptoethanol) and denatured by boiling at100° C. for 5 min. This step was repeated a total of three times.Samples were run on 15-18% polyacrylamide gels (SDS-PAGE) at a constantvoltage of 100 V using MiniProtein system (BioRad Laboratories). Theseparated proteins in the electrophoresis gel were stained using silverstaining.

The intact PWG gliadin evaluated by 1D gel revealed intense bands ofalpha, beta and gamma gliadin (MW=33-45 kDa) and weak bands of omegagliadin (MW=50-67 kDa) (Eckert et al., 2006 J Cereal Sci, 43:331-341).Digestion of these proteins mediated by pepsin (gastric digestion)resulted in the formation of smaller peptide fragments below 25 kDa.Sequentially, trypsin and chymotrypsin digestion generated smallerpeptides (less than 15 kDa), resulting from the hydrolysis processmediated by these enzymes (FIG. 1A).

In order to verify whether PWG gliadin peptides obtained by the processof gastrointestinal digestion were recognized by the anti-33-merantibody, a Western blot with this antibody was carried out for thesamples described above: undigested PWG gliadin, PWG gliadin aftergastric digestion and PWG gliadin after intestinal digestion (priorgastric digestion). Protein extracts initially obtained were separatedby SDS-PAGE and then incubated with G12 antibody onto PVDF membranes.After that, the samples were incubated in blocking buffer (TBS with 5%skim milk) overnight, after G12 antibody was added (1:5000 dilution inblocking solution). After 3 washes, membranes were incubated withsecondary antibody anti-mouse IgG conjugated to phosphatase (Sigma, St.Louis, Mo.) (1:2000 dilution in blocking solution). The membrane wasdeveloped using Sigma-Fast system.

The G12 antibody was able to recognize the different factions into PWGgliadin. After gastric digestion, peptide fragments formed remainedbeing recognized by the G12 antibody (FIG. 1B). Sequential treatmentwith pancreatic enzymes (trypsin, chymotrypsin) resulted in the presenceof smaller peptides also recognized by moAb G12.

In order to determine the capacity of the G12 antibody to quantify thetoxic peptides generated, the concentration of 33-mer and analoguespeptides obtained after gastrointestinal simulation of PWG gliadin wasdetermined by competitive ELISA also using the G12 antibody. Thecompetitive ELISA is a very suitable technique to monitor glutendigestion since it is capable of detecting both intact proteins andsmall protein fragments: the latter could be underestimated by sandwichELISA, because the detection of antigens requires at least two differentepitopes on the peptide molecule.

The relative amount of immunotoxic epitopes contained in the samples wasquantified by competitive ELISA using moAb G12-HRP (Biomedal SL,Seville, Spain). Maxisorp microtiter plates were used for this assay(Nunc, Roskilde, Denmark), which were coated with 100 μL/well of Sigmagliadin solution (5 μg/mL) in 0.1 M PBS (Na₂CO₃—NaHCO₃, pH 9.6), andincubated at 4° C. overnight. The plates were washed with PBS 0.05%Tween® 20 and blocked with blocking solution (PBS, 5% skimmed milk) for1 h at room temperature. Serial dilutions of the standard (gliadin or33-mer peptide) and samples studied were made in PBS with 3% BSA (100μL) and 100 μL of moAb G12 conjugated to HRP solution was added to eachone (1:10,000 in PBS with 3% BSA). Samples were pre-incubated 1 h atroom temperature with gentle agitation, and then added to the wells.After 30 minutes of incubation, samples were washed, and 100 μL/well ofsubstrate solution was added (TMB, Sigma, St. Louis, Mo., USA). After 30minutes incubation at room temperature in darkness, the reaction wasstopped with 1M sulfuric acid (100 μL/well), and absorbance was measuredat 450 nm (UVM340 microplate reader, Asys Hitech GMBH, Eugendorf,Austria). Gliadin/33-mer concentration was determined using the4-parameter model.

The concentration of 33-mer both in intact PWG gliadin and subjected togastric and intestinal digestion was determined by this method. Gastricdigestion of PWG gliadin resulted in a slight increase in the levels oftoxic peptide. This increase is probably due to the opening of themolecules constituting the gliadin fractions so that the epitopes ofanti-33-mer present are more accessible, and thus, can be identifiedwith greater specificity (intact PWG gliadin=21, 6 ng 33-mer/μg vs. PWGgliadin after gastric digestion=24.5 ng3 3-mer/μg). After the followingprocess of intestinal digestion the moAb G12 continued to recognize thePWG gliadin peptides formed, although with less specificity (7.5 ng33-mer/μg) (FIG. 1C).

In contrast to these results, both in vitro and in vivo studies madewith the 33-mer peptide demonstrate the great stability of this peptideto rupture by gastric, pancreatic and intestinal endoproteases. Itsfeatures make it to be suggested as the main promoter of theinflammatory response to gluten in celiac patients (FIG. 2A) (Shan etal., 2002, Science, 297:2275-2279, 2005, J Proteome Res 4:1732-1741).

To verify that the 33-mer peptide remains intact after gastricproteolysis (mediated by pepsin) and sequential intestinal proteolysis(mediated primarily by trypsin and chymotrypsin) an in vitro simulationof gastrointestinal digestion of this peptide was performed. Theconcentrations of 33-mer peptide obtained after each of the digestionprocesses were determined by competitive ELISA using the anti-33-mermonoclonal antibody. The concentration of 33-mer obtained after gastricdigestion did not differ significantly with respect to the non-digestedpeptide (194 μg/mL vs. 186 μg/mL, respectively, p=0.4469). Equally,exposure of the 33-mer to the enzymes trypsin and chymotrypsin(intestinal digestion), did not change the levels of this peptide incomparison with untreated peptide (169 μg/mL vs. 186 μg/mL,respectively, p=0.1024) (FIG. 2B). These results confirm the highstability of the 33-mer to hydrolysis by enzymes involved in thedigestive process.

The results obtained by ELISA were confirmed by Western blot analysis.Tricine-SDS-PAGE and Western blot were performed under standardconditions (Sousa et al., 2001, Mol Cellular Biol, 7:204-213). Theimmunoblotting showed bands of approximately 3.5 kDa in the samplecontaining unprocessed 33-mer as well as in that containing 33-mersubject to gastrointestinal digestion (theoretical 33-mer molecularweight 3.9 kDa, PIR, Protein Information Resource, Georgetown UniversityMedical Center, USA) (FIG. 2C).

Similarly, the ability of moAb G12 to detect hydrolysates was assessedusing a system for rapid detection of gluten, immunochromatographicstrips based on G12 moAb (GlutenTox stick, Biomedal S.L.). The detectionlimit for gliadin and hydrolyzed gliadin was 30 ng/ml (6 ppm of gluten)and 50 ng/ml (10 ppm of hydrolyzed gluten), respectively, while for the33-mer peptide and 33-mer peptide after digestion was 0.5 ng/mL in bothcases. These results suggest that the analysis method is highlysensitive for both the intact proteins/peptides and their respectivehydrolysates.

The results obtained by Western blot, competitive ELISA andimmunochromatographic strips suggest that anti-33-mer G12 antibody couldbe used to monitor the presence of toxic gliadin peptides and othergluten prolamins during the digestive process. At least one third ofpeptides reactive for G12 remained resistant to gastrointestinaldigestion. Therefore, a substantial portion of prolamins epitopes ofingested food that were detected with moAb G12 may be resistant togastrointestinal digestion and their detection may be appropriate in thegastrointestinal tract.

EXAMPLE 2 Detection and Semiquantification of Gluten Proteins/Peptidesin Feces of Healthy Individuals Undergoing Gluten Controlled Diet

The present example shows how the digestion that gluten proteins sufferin vivo in healthy individuals occurs, and also to determine the abilityof moAb G12 to detect these proteins/peptides excreted through thefeces. An assay was carried out in which the type and quantity of glutenconsumed in healthy individuals was controlled (n=11, 7 men and 4 women,mean age 24-42 years). The inclusion criteria were the absence ofdiseases, gastrointestinal symptoms, medications, antibiotics in thelast two months and no family history of CD. All participants wereassessed for CD, showed normal serum tTG levels and HLA-DQ phenotype wasnot DQ-2/-8. Hemoglobin levels and blood biochemical analysis, includingkidney and liver tests were within normal values. The local ethiccommittee from “Hospital Universitario de León” approved this study andinformed consent was obtained from the subjects.

For this study the following protocol was adopted:

-   -   Diet:        -   The subjects were instructed to follow a diet in which the            type and amount of gluten consumed was controlled within 15            days of this study. First, the subjects consumed a strict            gluten-free diet for a week. The following 4 days, 9 g of            unprocessed gluten were ingested, distributed in three meals            a day. In the last 4 days, the dose was increased to 30 g of            gluten, similarly distributed.    -   Fecal Sampling:        -   Fresh feces were collected from 11 subjects who participated            in the study under different diet conditions: normal diet,            GFD, GFD+9 g of gluten and GFD+30 g of gluten. The sampling            was made before GFD and after each of the diets tested. All            samples were homogenized and aliquoted within 3 hours after            defecation.    -   The extraction of prolamins from feces and gliadin solution:        -   Prolamins were extracted by mixing 1 g of feces with 10 mL            of ethanol 60% (v/v) on a rotary shaker for 1 h at room            temperature. The suspension was centrifuged at 13,000×g for            10 min and the supernatant was removed. The positive            control, PWG gliadin, was also prepared in ethanol 60% (v/v)            at 1 mg/mL.        -   First, fecal samples was collected from individuals            analyzed, who maintained a normal diet in which gluten was            present (bread, pasta, cookies, etc.). The presence of            gluten polypeptides in fecal extracts was determined            semiquantitatively using immunochromatographic strips based            on G12 antibody, in serial dilutions of the sample to            represent a wide range from less than 6 ppm to over 500 ppm.            Samples were diluted (1:10 to 1:20,000) in the dilution            solution proposed by the manufacturer (it was tested for 6,            25, 50, 100, 250 and 500 ppm of gluten)            Immunochromatographic strips were immersed in the different            samples (300 μL) for 10 min and allowed to air dry. In this            case, all individuals showed an excretion of gluten            proteins/peptides in feces with values above 500 ppm (FIGS.            3A and 3B).

Once confirmed the feasibility of the method for the detection of glutenin feces, the correlation between the amount of gluten consumed andamount of gluten excreted were tested. For this, the 11 subjectsfollowed a controlled diet of gluten. First, these individuals consumeda strict gluten-free diet for one week, then they ingested 9 g of glutenper day divided into the main meals for a period of 4 days (taking intoaccount the filling time of the large intestine) and finally, theyconsumed 30 g of gluten per day, equally divided into the main meals fora period of 4 days. In order to avoid differences in the measurement dueto the ingestion of different gluten products with different origin, inall cases it was administered the same type of gluten (without heattreatment). The proposed schedule took into account that in healthypeople the transit time is 45±16 hours (mean ±standard deviation) with adiet rich in fiber and over 70 hours on low fiber diets (Stasse-Wolthuiset al., 1979, Am J Clin Nutr., 32:1881-1888).

Fecal samples collected during the period in which the individualfollowed a gluten-free diet showed, in all cases, gluten levels belowthe detection limit of the method (6 ppm of intact gluten, 10 ppm ofhydrolyzed gluten). In contrast, when there was a 9 g intake of glutenper day it was found that the amount of gluten detected was above 250ppm in all samples, except one which had values between 6 and 25 ppm.When individuals consumed 30 g of gluten per day the levels of glutenexcreted were above 500 ppm (FIGS. 3A and 3B), more than 100 timesgreater than the detection limit of the method. Therefore, there is acorrelation between the amount of gluten consumed and the amount ofpeptides with G12 epitopes excreted in feces.

In order to demonstrate the suitability of moAb G12 in the detection ofgluten proteins/peptides excreted in feces, protein extracts obtained bytreatment with 60% ethanol as well as controls, PWG gliadin and gluteningested by subjects, were separated by SDS-PAGE. After that theproteins/peptides were stained with silver staining or transferred to amembrane and analyzed by Western blot with moAb G12 (FIGS. 3C and 3D).The results indicated that the moAb G12 reacts with samples derived froma conventional uncontrolled diet, GFD+9 g of gluten and GFD+30 g ofgluten, as well as the positive controls, PWG gliadin and ingestedgluten. However, in the sample derived from a GFD no peptides/proteinswere found in feces (FIG. 3D).

EXAMPLE 3 In Vivo Monitoring of Gluten Immunotoxic Peptides in Fecesfrom Individuals Following a Controlled Diet with Gluten

This example shows how the partial digestion of reactive peptides can bedetermined by ELISA with the G12 anti gliadin 33 mer antibody, asconvenient method, due to its simplicity, sensitivity and economy. Inthe case of the detection of proteins/peptides from gluten, sandwichELISA systems are designed to quantify intact proteins but mayunderestimate hydrolyzed gluten. Gluten passage through thegastrointestinal tract results in the hydrolysis of the majority of it:a competitive ELISA is able to quantify toxic peptides, even at thelevel of a few amino acids, so it would be a convenient method forquantification.

Therefore, the aim of this study was to determine the concentration oftoxic peptides present in the feces from healthy individuals by G12competitive ELISA using as standard curve 33-mer peptide. Eachexperiment was carried out in triplicate on separate days. Allstatistical analysis was performed using SPSS software for Windows. Datawere expressed as mean, maximum, minimum and percentile values 25 and75. Differences between groups were examined using Friedman test andWilcoxon test for comparing two related samples. A statisticalprobability of p<0.05 was considered significant.

As in the previous test, the fecal samples analyzed were the samplescorresponding to the periods of intake: uncontrolled diet, GFD, GFD+9 gof gluten and GFD+30 g of gluten. Fecal samples collected during theperiod in which individuals followed a gluten-free diet had levels oftoxic peptide below the quantification limit of the method (5.4 pg33-mer/mg of sample). However, when they ingested 9 g of gluten per day,immunoreactive peptides were detected in feces in all cases, being inthe range between 3.49 and 9.62 ng 33-mer/mg of feces, 600 times higherthan the detection limit of the method. Finally, when individualsconsumed 30 g of gluten per day the levels of 33-mer obtained increasedin all cases, with respect to the period of ingestion of 9 g/day (6.69to 28.00 ng 33-mer/mg of feces, p=0.018, with respect to GFD+9 g) (FIG.4), more than 1,000 times higher than the detection limit of the method.These results agree with those obtained previously in the glutendetection in feces by immunochromatography. The method based on theanti-33-mer antibody could estimate the amount of gluten proteinsconsumed by measuring reactive peptides excreted in the feces, and theingestion of few grams of gluten per day could be detected in quantitiesexceeding 600 times the detection limit. Therefore, an intake of greaterthan 10 mg of gluten daily could be assumed as detectable byimmunological assays based on moAb G12.

The invention claimed is:
 1. A process for monitoring compliance ofgluten concentration in a gluten free diet comprising: (a) isolating afecal sample from a human ingesting a gluten free diet; (b) treating thefecal sample of step (a) with a hydroalcoholic solution, the treating ofthe fecal sample providing extracted immunotoxic gluten peptides; (c)contacting the extracted peptides of step b) with at least onemonoclonal antibody which specifically binds the gluten immunotoxicpeptides having epitopes of including any sequence selected from thegroup consisting of SEQ ID NO: 2 and SEQ ID NO: 6, the contacting of theextracted peptides with the at least one monoclonal antibody formingmonoclonal antibody-gluten peptide complexes; (d) detecting themonoclonal antibody-gluten peptide complexes formed in step (c); (e)quantitating the extracted immunotoxic peptides based on the detectionof at least one of the monoclonal antibody-gluten peptide complexes; and(f) monitoring the quantitated immunotoxic peptides of step (e), whereinan increase of the quantity of the extracted peptides above 160 ng/g offeces is indicative of non-compliance of the gluten concentration in thegluten free diet.
 2. The process for monitoring according to claim 1 inwhich step (d) is carried out by an indirect ELISA, a competitive ELISA,a sandwich ELISA, immunochromatographic strips,fluorescentimmunomicroparticles, magnetic immunoparticles, Western blot,electronic biosensors or resonance biosensors.
 3. The process formonitoring according to claim 1 in which the antibody of step (c) is amonoclonal antibody conjugated to an enzyme that allows a quantitativeassay using chromogenic, fluorogenic or luminescent substrates.
 4. Theprocess for monitoring according to claim 1, in which step (d) comprisesquantifying the detected complex by means of a reference peptideselected from the group consisting of SEQ ID NO: 2 and SEQ ID NO:
 6. 5.The process for monitoring the according to claim 1, in which step (d)is carried out by an immunochromatographic strips of rapid detection. 6.The process according to claim 1, further comprising providing beforestep (a) a kit containing: the hvdroalcoholid solution of step b) forthe extraction of gluten peptides in feces, a reference peptide standardcomprising at least one of the immunogenic peptide selected from thegroup consisting of SEQ ID NO: 2 and SEQ ID NO: 6; and the at least onemonoclonal antibody of step (c).